Fibrosing diseases are known to affect many different mammalian organs. Common examples include the kidney (glomerulonephritis), bladder, prostate (benign prostate hypertrophy), lung (emphysema) and liver. But essentially all tissues are affected, in one way or another, by the fibrotic process. This is due mainly to the wide range of different cells types, such as fibroblasts, smooth muscle cells, and even epithelial cells, that are involved in fibrotic disease. The common thread that links all these cells types with fibrotic disease is the synthesis of connective tissue.
Until recently, it was believed that fibrosis was a terminal and irreversible process consisting of the deposition of intert connective tissue in the scarring process. It is now thought that fibrosis is a dynamic process up to the end stages of disease. In other words, deposition of scar tissue continues until the affected tissue is almost completely replaced by scar tissue. This finding has considerable therapeutic import since even a fairly late diagnosis of the disease may permit an effective implementation of therapy. It remains quite important to make an accurate diagnosis as early as possible, however, in order to minimize the damage to affected tissues. Unfortunately, overt clinical signs of disease, usually marked by the beginning of organ failure, often do not occur until more than one-half of the organ has been scarred.
One of the devastating manifestations of fibrotic disease is kidney failure. In fact, the most common cause of end-stage renal disease in humans is the result of gradual, glomerular scarring known as glomerulosclerosis. United States Renal Data System, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, 1991. The glomerulus is a tuft of capillaries situated at the origin of the vertebrate kidney that is responsible for filtering impurities from the blood, resulting in the formation of urine. A major cause of glomerular scarring is fibrosis resulting from the excessive deposition of extracellular matrix (ECM) components in the glomerular region. Presumably, this deposition results from a deviation in the tightly regulated balance between the synthesis and degradation of the molecules which comprise the ECM. Little is known concerning the molecular basis of this abnormality.
The ECM is a complex network of macromolecules that fills the tissue space between cells. Until recently, it was thought that the ECM provided a relatively inert scaffold on which cells found support. But it is now clear that this structure is intimately involved with the development and function of many cell types.
The ECM of vertebrates is made up of two general groups of molecules, glycosaminoglycans (GAG's) and fibrous proteins. GAG's are long polymers of repeating disaccharide units, most of which are covalently linked to protein molecules to form proteoglycans. The fibrous proteins are of two classes, adhesive and structural. The major fibrous protein, collagen, is of the latter type.
There are at least ten different collagens presently known, all comprised of trimeric helices. The individual protein subunits that comprise the helix are characterized by a repeating "gly-X-Y" unit, where X and Y can be any amino acid, but are often proline. The small glycine residue allows for tight winding of the three subunits into a triple .alpha.-helix structure. The best characterized proteins of this family of molecules are collagens I-IV. Type II collagen and type III collagen are each composed of three identical subunits, .alpha.1(II) and .alpha.1(III) respectively. Type I collagen is comprised of two .alpha.1(I) subunits and one .alpha.2(I) subunit. Likewise, type IV collagen is comprised of two .alpha.1(IV) subunits and one .alpha.2(IV) subunit. Depending on the tissue type, the collagen make up of the ECM can differ dramatically and individual collagens may be further modified depending on their location and role.
Type IV collagen has been shown to be the major component of the glomerulosclerotic lesion. Morel-Maroger Striker et al., Lab. Invest. 51:181-192 (1984). Type I collagen, which is not normally found in the glomerular ECM, has also been identified by immunofluorescence in the sclerotic lesion. Merritt et al., Lab. Invest. 63:762-769, 1990. In addition, there is evidence linking most other kinds of collagen with the generation of scar tissue. Id.
Yet another group of molecules which contribute to the extracellular matrix system, and potentially to fibrotic disease, is the metalloproteinase family. These enzymes mediate matrix degradation by type-specific cleavage of collagens. Liotta, L. A. and W. G. Stetler-Stevenson, Sem. Can. Bio. 1:99-106 (1990); Woessner, J. F., FASEB J. 5:2145-2154 (1991). For example, interstitial collagenases cleave type I and type III collagen, whereas 66-72 and 92 kDa gelatinases degrade non-helical type IV and V collagens as well as denatured interstitial collagenases. The action of these enzymes is modulated by a family of tissue inhibitors of metalloproteinases, or TIMP's, two of which, TIMP-1 and TIMP-2, have been characterized in certain cells and tissues from humans. Carmichael et al., Proc. Nat'l Acad. Sci. 83:2407-2411 (1986); Stetler-Stevenson et al., J. Biol. Chem. 264:17374-17378 (1989). These inhibitors inactivate all matrix metalloproteinases through formation of an enzyme-inhibitor complex exhibiting a 1:1 stoichiometry. Mesangial cells, one of the three major cell types in the glomerulus, synthesize both TIMP-1 and TIMP-2, as well as a variety of metalloproteinases. Martin et al., J. Immunol. 137:525-529 (1986); Kawanishi, et al., J. Am. Soc. Nephrol. 2:577 (1991).
The overall gross pathology of fibrotic disease is characterized by an increase in tissue rigidity, a concomittant loss of elasticity, and eventual replacement of organ tissue with scar. Such alterations also adversely affect the function of the organ. The specific cellular changes that occur are presently the subject of intense investigation. What can be said is that the deposition of excess ECM, which leads to scar formation, causes substantial changes in the behavior of cells. The reasons for the production and deposition of excess ECM remain largely unknown.
Unfortunately, the study of the phenomena discussed above has been hampered for a number of reasons. Glomeruli represent only a small fraction of the kidney, thus making studies of whole kidney, or even cortex, difficult to relate to glomerlular change. In addition, it has not been possible to obtain sufficient quantities of such tissues from human subjects in order to do meaningful research. Also, glomeruli appear to be regulated independently of other kidney tissue, making studies of whole kidneys difficult to relate to these specific renal tissues. Doi et al., Am. J. Pathol. 131:398-403 (1988); Pesce et al., Lab. Invest. 65:601-605 (1991). Study of collagen synthesis is further slowed by low levels of messenger RNA's corresponding to these proteins. Laurie et al., J. Cell Biol. 109:1351-1362 (1989). And finally, while glomerular cells can be propagated in vitro, such studies cannot be readily extrapolated to the intact structure since phenotypic changes in matrix metabolism occur in cell culture. Striker et al., Transplant. Proc. 12:88-99 (1980); Morel-Maroger Striker et al., Lab. Invest. 51:181-192 (1984). As a general matter, these difficulties are characteristic of the study of fibrotic disease in other tissues as well.
The foregoing serves to highlight the lack of adequate diagnostic capability with respect to glomerular fibrosis. Without a simple and effective way to detect early fibrotic changes, there is little hope that treatment can be implemented soon enough to avoid tissue damage and possible organ failure.